This research seeks to identify and characterize the hierarchy of regulatory phenomena which govern the expression of a simple metabolic pathway in a eukaryotic organism. The pathway is that of nitrate assimilation in Neurospora crassa, which consists of two pyridine nucleotide-dependent electron transfer enzymes: nitrate reductase and nitrite reductase. The expression of both enzymes of this pathway is induced by the presence of nitrate or nitrite, and both are repressed by the end product of the pathway, ammonium, or by primary products of ammonium assimilation such as glutamate or glutamine. This latter effect, which is termed nitrogen metabolite repression, is dominant to nitrate induction. Extensive mutational analysis of the genetics of nitrate assimilation in Neurospora has revealed at least 8 genes, the nit genes. Two of these genes, nit-3 and nit-6, are structural genes encoding the enzymes, nitrate reductase and nitrite reductase, respectively. At least 4 genes, nit-1,7,8 and 9, function in the production of the molybdenum cofactor essential to the nitrate reductase. Two genes serve regulatory roles: nit-4/5, a single locus, is required for induction of the nitrate assimilation pathway in the presence of nitrate, whereas nit-2 is involved in nitrogen metabolite repression. This nit-2 gene has a pleiotropic role, affecting a number of metabolic processes which, like nitrate assimilation, provide for nitrogen nutrition in the absence of exogenous ammonium, e.g., purine catabolism of urea degradation. Apparently the nit-2 gene product is a positive gene regulator necessary for the expression of various alternative pathways of nitrogen acquisition. The hypothesis under active investigation here is that glutamine is the key metabolite signalling cellular nitrogen sufficiency and that it acts indirectly, through changes in glutamine synthetase, to halt nit-2 gene expression and thereby manifest nitrogen metabolite repression.